Research ArticleRat Bone Mesenchymal Stem Cell-Derived Exosomes Loaded withmiR-494 Promoting Neurofilament Regeneration and BehavioralFunction Recovery after Spinal Cord Injury

Wei Huang ,1,2 Miaoman Lin ,1 Cunheng Yang ,1 Fumin Wang ,1 Meng Zhang ,1

Junxiao Gao ,1 and Xiaobing Yu 1

1Department of Orthopaedics, Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, China2Department of Orthopaedics, Dongguan Tungwah Hospital, Dongguan 523000, China

Correspondence should be addressed to Xiaobing Yu; yuxiaobing1976@hotmail.com

Received 14 June 2021; Revised 5 September 2021; Accepted 9 September 2021; Published 1 October 2021

Academic Editor: Dragan Hrnčić

Copyright © 2021 Wei Huang et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Exosomes (Exo) exhibit numerous advantages (e.g., good encapsulation, high targeting efficiency, and easy to penetrate theblood-brain barrier to the central nervous system). Exosomes are recognized as prominent carriers of mRNAs, siRNAs,miRNAs, proteins, and other bioactive molecules. As confirmed by existing studies, miR-494 is important to regulate theoccurrence, progression, and repair of spinal cord injury (SCI). We constructed miR-494-modified exosomes (Exo-miR-494). Asindicated from related research in vitro and vivo, Exo-miR-494 is capable of effectively inhibiting the inflammatory responseand neuronal apoptosis in the injured area, as well as upregulating various anti-inflammatory factors and miR-494 to protectneurons. Moreover, it can promote the regeneration of the neurofilament and improve the recovery of behavioral function ofSCI rats.

1. Introduction

Spinal cord injury (SCI) refers to a catastrophic event inspinal surgery, commonly leading to severe neurologicaland sensory dysfunction [1]. It still lacks effective treatmentmeasures in clinical practice, imposing huge psychologicaland economic burden to patients and the country’s medicalservices. Over the past few years, the powerful proliferationand differentiation potential of mesenchymal stem cells(MSCs) has achieved new progress for treating SCI [2].However, the transplanted MSCs have numerous defects(e.g., low survival rate, carcinogenicity, and poor targeting)[3]. As proven by numerous studies, the therapeutic effectof MSCs may be primarily attributed to the secreted exo-somes (Exo) [4].

Exos are small membrane vesicles originating fromintracellular corpuscles, exhibiting a diameter of about 30–200nm, which are packed with various proteins, lipids, andRNA. These vesicles can be released into extracellular fluid,

blood, and cerebrospinal fluid by cells of all living systems[5, 6]. Under physiological and pathophysiological condi-tions, they are capable of transferring their cargo betweensource cells and target cells via extracellular fluid, blood,and cerebrospinal fluid, and they critically impact intercellu-lar communication [7]. Compared with the transplantedMSCs, MSCs-Exo exhibit the advantages of long stay timein the body, low carcinogenicity, and high targeting effi-ciency, as well as low immune rejection and easy of passingthrough the blood-brain barrier. Thus, it acts as an excellentcarrier of foreign small molecule drugs.

Exosomes are closely related to the occurrence, progress,and recovery of central nervous system injury [8–10]. Afterspinal cord injury, exosomes can regulate the autonomicregeneration function of neurons and, also, affect theremyelination of the spinal cord, the metabolic activity,and inflammatory environment around the lesions. Exo-somes can also actively mobilize the internal regenerationability of neurons and reduce the adverse factors of the

HindawiOxidative Medicine and Cellular LongevityVolume 2021, Article ID 1634917, 15 pageshttps://doi.org/10.1155/2021/1634917

external surrounding microenvironment [11]. Studies haveshown that bone marrow mesenchymal stem cell-exosomescan protect the injured spinal cord by inhibiting pericytepyroptosis [12]. Furthermore, nanoscale exosomes secretedby stem cells have great potential in reducing apoptosis andinflammation, enhancing angiogenesis, and improving func-tional and behavioral recovery after spinal cord injury [13].

MicroRNAs (miRNAs) are small noncoding RNAmolecules, capable of regulating various biological activitiesand profoundly impacting cell proliferation, differentiation,aging, death, neural activity, etc. miRNAs show a close rela-tionship to the pathological processes of spinal cord injury(e.g., inflammatory reaction, demyelination, oxidative stress,and neuronal apoptosis) [14].

A large number of miRNAs have been found in the cen-tral nervous system of mammals, which play an importantrole in neurogenesis, development, and regeneration [15].As reported by Xu et al., in the treatment of spinal cordinjury, the overexpressions of miR-124 and miR-133b pro-mote the differentiation of neural stem cells [16, 17]. It hasbeen reported that miR-494 can reduce autophagy andapoptosis of neurons after SCI by targeting IL-13 [18]. Inaddition, Wang et al. suggested that miR-494 is capable ofregulating the effects of the Nogo/NgR signaling pathwayon astrocyte proliferation and synaptic remodeling afterSCI [19].

Though miR-494 exhibits a strong repair ability for SCI,the amount of secretion by living cells is low, and the effi-ciency of in vivo transportation is limited, thereby adverselyaffecting its therapeutic effect. This study planned to loadexogenous miR-494 into exosomes through reagent transfec-tion to determine whether exosomes can effectively delivermiR-494 to the spinal cord lesion, as well as verify the repaireffect of ExomiR-494 on SCI rats.

2. Methods and Materials

2.1. Experimental Design. The whole experiment is dividedinto three parts, “construction of ExomiR-494/Exo-miR-494,”“in vitro research,” and “in vivo research.” As shown inFigure 1, firstly, we used the kit to extract the exosomes fromrat MSCs and then designed and synthesized the miR-494mimics according to the gene sequence of miR-494. Next,we used the Exo-Fect™ transfection kit to transfect miR-494 into the MSCs-Exo and got the ExomiR-494. In the secondpart, we cocultured the constructed Exo-miR-494 with dor-sal root ganglions (DRGs) and rat NR8383 macrophages inthe spinal cord injury environment to study whether it hasantiapoptotic and anti-inflammatory effects. Finally, we con-ducted animal experiments. We injected ExomiR-494 into SCIrats by tail vein injection to further study its effects on motorand behavioral functions of SCI rats.

2.2. Animals. The experimental procedures were approvedby the Affiliated Zhongshan Hospital of Dalian UniversityScience Review Committee and Animal Research EthicsReview Committee and conducted in line with the NationalInstitutes of Health Guide for the Care and Use of Labora-tory Animals.

First, we will use 2-week-old SD rats to extract boneMSCs. Then, in order to ensure that the grouping is statisti-cally significant, we also need 6 SCI rats for the in vitroorgan imaging experiment. Next, we need 6 normal healthyrats and 6 SCI rats for the drug toxicity test (the same animalcan take the lung and liver at the same time, as well as thedetermination of lactate dehydrogenase, superoxide dismut-ase, and alanine aminotransferase in the liver).

Next, we carried out the animal experiment to verify thetherapeutic effect of ExomiR-494 on spinal cord injury. On the

MSCs Exo

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MSCs-Exo miRNA-494 mimic

MiRNA-494 genesequences from

NCBI library

Chemical transfectionby Exo-Fect Kit

SCI ratsCells in the environment of SCI

Figure 1: The flow chart of the whole experiment.

2 Oxidative Medicine and Cellular Longevity

whole, grouping of animal experiments, i.e., the Controlgroup (rats were treated with PBS after SCI), Sham group(rats were treated with PBS after sham operation), Exo-miR-con group (rats were treated with pure exosome afterSCI), and Exo-miR-494 group (rats were treated with Exo-miR-494 after SCI). In order to ensure the statistical signifi-cance of each experiment and ensure that the number ofrats in each group is 6, we divided the animals into the Con-trol group (n = 6), Sham group (n = 6), Exo-miR-con group(n = 6), and Exo-miR-494 group (n = 6) to form a largegroup. In this way, we need a large group of animals todetermine the relative expression of miR-494 and growth-associated protein-43(GAP-43) in vivo. Similarly, we needa large group of animals for the hematoxylin-eosin (HE)experiment, immunofluorescence analysis experiment, loco-motor capacity assessment experiment, Paw WithdrawThermal Latency (PWTL) experiment, and hind leg neuro-physiological experiment. Statistically, we need 2 2-week-old SD rats, 6 normal healthy rats, 42 sham rats, and 138SCI rats (excluding the rats with SCI model failure or death,continue to supplement the above conditions, and all animalnumbers are confirmed by the Animal Research EthicsReview Committee).

2.3. Extraction and Identification of Bone Marrow MSCs.Firstly, male SD rats (80-100 g) of 2 weeks old were pre-pared, the whole bone marrow culture method was used toextract MSCs, and then, MSCs were cultured in primary cul-ture until the cell density reached 90%. Subsequently, theserum-free FBS (Gibco™, USA) was used for 48h expansionculture in the complete medium to obtain about 300mL ofstem cell culture supernatant for subsequent experiments.

Next, we used flow cytometry to identify the expressionof the bone marrow MSC marker protein. In short, firstly,MSCs were digested with trypsin and mixed with 100μLPBS, and then, 10μL of CD29, CD45, and CD90 antibodies(eBioscience, USA) was added. The mixture was wrappedwith tin foil and incubated at 4°C for 30min. After incuba-tion, centrifuge for 5min, clean 3 times, and resuspendMSCs with 500μL PBS. Finally, the marker protein wasdetected by flow cytometry (Cytek, USA), and the data wereprocessed by FlowJo 8.7 software.

2.4. Preparation and Identification of Exosomes Derived fromBone Marrow MSCs. With the kit method, the exosomeswere separated from the collected cell supernatant. In brief,the cell supernatant was placed at 4°C and then centrifugedat 3000 g for 10min to remove excess cell debris. Subse-quently, it was centrifuged to remove impurities. GS™ Exo-some Isolation Reagent (for serum or plasma, GeneseedBiotech Co., China) was added to the supernatant, and theinstructions (Supplement 1) were complied with to extractexosomes from the extracellular supernatant. In summary,500μL of the rat supernatant was extracted, and 1/3 timesthe volume of the supernatant was added to the solutionA. And the mixed solution was left to stand at 4°C for15min. At ambient temperature, centrifugation was con-ducted again at 13,000 g for 10min, and the supernatantwas aspirated; the solution was centrifuged at 13,000 g for

30 s, and the residual supernatant was aspirated. Lastly, theidentical volume of B solution was added as Reagent A,and the solution was mixed by pipetting and centrifugedat 13,000 g. Finally, the supernatant was aspirated andremoved; afterwards, we used bicinchoninic acid (BCA)to detect the protein concentration in the exosome and thenresuspended it in PBS with a total protein concentration of10μg/μL [20]. The resulting precipitate was an exosome,which was stored at -80°C. The mentioned method wasrepeated to extract all the exosomes in the supernatant of500mL for subsequent cell and animal experiments.

Next, we used the followingmethods to identify exosomes.

2.4.1. Transmission Electron Microscopy (TEM). Take out5μL exosomes, add 5μL PBS, then add the mixture to thecopper wire, and stand for 1min; next, 10μL phosphotung-stic acid was added to the copper mesh and stood for 1minto remove the liquid suspended on the surface (refer toSupplement 2 for specific methods). Finally, the imagingwas detected by 80 kV transmission electron microscopy(Hitachi, JAPAN).

2.4.2. Nanoparticle Tracking Analysis (NTA). Take 10μLexosomes and add 990μL PBS which was mixed evenly,and nanoparticle tracking analysis instrument (ParticleMetrix, Germany) was used to detect the sample. Besides,we detected the marker proteins CD9 and CD63 of exo-somes by western blot (WB) analysis (see “WB Analysis”for specific methods).

2.5. Preparation of miR-494 Mimic and Negative Controls(NC). By complying with the instructions, miR-494 mimicsand miR-494 NC were synthesized by Invitrogen (Gene-Pharma, Shanghai, China), treated with diethyl pyrocarbo-nate (DEPC), and then placed and stored at a lowtemperature and in a dark place. The oligonucleotides usedin this sequence are shown in Table 1.

2.6. MiR-494 Was Loaded into Exosomes by ChemicalTransfection. As mentioned above [21], we used Exo-Fect™(System Biosciences, Palo Alto, USA) to load miR-494. First,miR-494 (100-300 pmol), exosomes (200μg in 50μL), andExo-Fect were mixed and incubated at 37°C for 20min (forthe Control group, only miR-494 and exosomes were mixedwithout adding Exo-Fect), then ExoQuick-TC reagent (Sys-tem Biosciences, Palo Alto, USA) was used to culture themixture on ice for 20min. Finally, exosomes/ExomiR-494 werecentrifuged at 13,000 × g for 5min to collect and resuspendin phosphate buffer saline (PBS, Gibco™, USA) for reversetranscription (qPCR).

2.7. Quantitative Determination and AntidegradationTest of ExomiR-494

2.7.1. ExomiR-494 Loading Capacity. 5′-32P were used to labelmiR-494. The loading capacity of exosomes was determinedby the radioactivity in the supernatant and then quantifiedwith a Packard instant imager. The loading rate was calcu-lated using the following equation: themiR − 494 loadingrate = ðCPM in pellet/CPM in supernatantÞ × 100. Moreover,

3Oxidative Medicine and Cellular Longevity

the loading capacity of ExomiR-494 was calculated by theaforesaid formula.

2.7.2. ExomiR-494 Antidegradation Test. ExomiR-494 were incu-bated with 500μL RNase A with various concentrations(0.01μg/mL, Solarbio, USA). After being incubated at 37°Cfor 0, 30, 60, and 90min, naked miR-494 mimic was classi-fied as the control. Moreover, the undegraded miR-494 wasquantitatively analyzed by RT-PCR.

2.8. Apoptosis Test. Rat dorsal root ganglion cells (DRGs)were obtained from the cell bank center of the ShanghaiAcademy of Biological Sciences (SIBS) and were seeded ina 6-well plate with 1 × 106 cells/well. When DRGs fusedmore than 90%, the SCI environment was simulated with10mg lipopolysaccharide (LPS, YT1319, YITA, China) andhypoxia (2% O2) for 24h. Referring to previous studies[20], next, the experimental groups were as follows (n = 3):Control group (0.5mL PBS), Exo-miR-Con group (100μgpure exosomes in 0.5mL PBS), and Exo-miR-494 group(100μg ExomiR-494 in 0.5mL PBS). After 24 hours of cocul-ture, DRGs were washed twice with PBS, and the survivalof the cells was evaluated by an acridine orange endotheliumbromide (AOEB) staining kit (Jizhi Biochemical Technol-ogy, Shanghai), and apoptotic proteins (Caspase-3, Bax,and Bcl-2) were also extracted for western blotting as previ-ously described [22].

2.9. Enzyme-Linked Immunosorbent Assay (ELISA). Theculture, seed plate, and grouping of rat NR8383 macro-phages (ATCC, SIBS, China) were the same as DRGs. Theexperimental groups were as follows (n = 3): Control group(0.5mL PBS), Exo-miR-Con group (100μg pure exosomesin 0.5mL PBS), and Exo-miR-494 group (100μg ExomiR-494

in 0.5mL PBS). After 48 hours of coculture, the expressionof inflammatory factors was detected by ELISA kits: ratTNF-α, IL-6, IL-8, and IL-10 ELISA kit (Thermo Scientific,Shanghai, China).

2.10. Establishment of SCI Rat Model and ExperimentalGrouping. The rats were anesthetized with 4% sodium pen-tobarbital (50mg/kg; Beyotime Biotechnology, China). Atthe T10 level, a 2 cm incision was achieved to expose thespinal skeletal muscle in a sterile environment. Except forthe Sham group, the T10 segment was impacted by the spi-nal cord injury percussion apparatus (Yuyan Instrument,China) in the other groups. The weight of the metal rodreached 25 g, and the height was 50mm. Subsequently,involuntary twitch and tail swing were identified in bothhind limbs of all rats, demonstrating that the injury reachedthe standard of the spinal cord injury model. In the Shamgroup, the above operations were performed without spinalcord injury. After suturing the wound, the rats after opera-

tion recovered in a warm incubator. In addition, rats in theSham group underwent the above operations without spinalcord injury.

After operation, the animals were urinated three times aday until they could urinate by themselves. Tail-vein admin-istration was performed per 24 h for 7 consecutive days afterestablishment of the SCI model. To be specific, the dosagewas as follows: Control group and Sham group were admin-istered with 1mL PBS, while the Exo-miR-con group with100μg pure exosomes (diluted in 1mL PBS), and the Exo-miR-494 group was treated with 100μg ExomiR-494 (dilutedin 1mL PBS).

2.11. ExomiR-494 Staining. According to one research [23], thedye working solution was prepared. In short, dilute the dyestorage solution with diluent C reagent (UR52303, Umibio,China), prepare the PKH67 dye working solution with theconcentration of 100nm, add 10μL PKH67 dye workingsolution to the ExomiR-494, mix well, and culture at roomtemperature for 5min. The reaction between ExomiR-494

and PKH67 dye working solution was terminated for 2minby adding 500μL 1% bovine serum albumin (BSA). After-wards, the volume of the mixture was added to 25mL andthen centrifuged for 45min at 120,000 g. The supernatantwas discarded, and the residue was suspended in PBS. Thefinal precipitate was PKH67-ExomiR-494.

2.12. Drug Toxicity Test of ExomiR-494 to the Lung and Liver.After 24 hours of treatment with ExomiR-494, the lung andliver tissues of rats in the Exo-miR-494 group were takenout for HE staining and compared with those of healthy rats.Simultaneously, the activities of lactate dehydrogenase(LDH), superoxide dismutase (SOD), and alanine amino-transferase (ALT) in the healthy rat liver were measured bythe enzyme labeling method. Firstly, we extract 20μg liverhomogenate, and PBS were used to lyse cells. Then, threeenzyme assay kits (Wanlei Bio, China) were added to the cellculture medium, the LDH activity, ALT activity, and SODactivity according to the instructions (Supplement 4, 5, 6).The following are the specific detection methods.

2.12.1. LDH Detection (Visible Light Colorimetry). First, take10-50μL rat liver homogenate. Then, add the LDH kit, mixwell, and place in a water bath at 37°C for 15min; finally,set the wavelength to 440 nm and the optical path to 1 cmand measure the absorbance OD value of each tube withan ultraviolet spectrophotometer (Shimadzu, Japan).

2.12.2. Calculation Formula of Tissue LDH Activity. 1.5% isproduced in the reaction system when each gram of tissueprotein reacts with the matrix at 37°C for 15min; 1μmolpyruvate is 1 unit (U).

2.12.3. SOD Detection (Hydroxylamine Method). First, take20μL rat liver homogenate. Then, add the SOD kit, mix well,and place at room temperature for 10min; finally, set thewavelength to 550 nm and the optical path to 1 cm andmeasure the absorbance OD value of each tube with anultraviolet spectrophotometer.

Table 1: Synthesized miR-494 mimics and negative control (NC).

Gene Sequence (5′ to 3′)miR-494 mimic UGAAACAUACACGGGAAACCUC

miR-494 mimic NC UUCUCCGAACGUGUCACGU

4 Oxidative Medicine and Cellular Longevity

2.12.4. Calculation of Total SOD Activity in TissueHomogenate.When the inhibition rate of SOD per mg tissueprotein in 1mL reaction solution reaches 50%, the corre-sponding SOD amount is one SOD activity unit (U).

2.12.5. ALT/GPT Detection (Visible Light Colorimetry). Mixthe ALT kit with 50μL rat liver tissue; place it at roomtemperature for 5min, wavelength 505nm, optical path1 cm, and zero with double distilled water; and measurethe absorbance OD value of each tube. Check the standardcurve to obtain the corresponding ALT/GPT activity unit.

Moreover, after 48 hours of administration, miR-494were extracted for further quantitative RT-PCR analysis.

2.13. Total RNA Isolation and Quantitative Real-TimePolymerase Chain Reaction (qRT-PCR). RNA extraction kits(Solarbio, China) were adopted to extract total RNA fromcells and spinal cord lesion tissues. To be specific, miRNAextraction kits (Solarbio, China) were used for miR-494 inexosomes or cells, target mRNA or miR-494 acted as tem-plates, and reverse transcription kits (Solarbio, China) wereemployed for reverse transcription into cDNA. Relativequantification of RNA expression levels was performed onan ABI 7500 Real-Time PCR system (Applied Biosystems,Carlsbad, CA). Furthermore, the 2-ΔΔCt method was employedfor relative quantification, and U6 was the internal referencegene (refer to Supplement 3 for specific methods). Table 2 listsall the primers applied in the experiment.

2.14. WB Analysis. RIPA buffer was employed to extract thetotal exosomes, protein of cells, and spinal cord tissue, andWB experiments were performed by complying with themanufacturer’s protocol. Primary antibodies include CD63,CD9, GAPDH, β-actin, Caspase-3, Bax, Bcl-2, and GAP-43(rabbit, Abcam, UK).

2.15. HE Staining of Spinal Cord Tissue. After 4 weeks ofadministration, rats in each group were anesthetized with4% sodium pentobarbital (50mg/kg). Then, 1 cm spinal cordsegments (including injury center) were stained with HEstaining. The center of the lesion was the most severetransverse section.

2.16. Immunofluorescence Analysis. After 4 weeks of admin-istration, the longitudinal spinal cords of rats in each groupwere cut into frozen sections at 0.02mm intervals. Subse-quently, the sections were permeabilized in 0.5% Tweensolution and blocked with 5% bovine serum, combined withmouse neurofilament (NF-H) antibody (1 : 1000, Abcam,UK) and rabbit glial fibrillary acidic protein (GFAP) anti-body (1 : 100, Abcam, UK) overnight at 4°C. Afterwards,the spinal cord sections were incubated with DAPI(1 : 1000, Invitrogen) and goat anti-rabbit IgG (H+L) sec-ondary antibody (1 : 300, Invitrogen) for 1 h, and the imagewas captured under a confocal fluorescence microscope(Leica Microsystems, Germany).

Furthermore, we use ImageJ software (V1.8.0.112) tomeasure the relative expression of NF and GFAP by averagefluorescence intensity. First, we use the ImageJ software toopen the picture of single fluorescence (NF or GFAP), then

adjust the threshold (software default), select the appropriatethreshold algorithm, and detect and analyze the result.The average fluorescence intensity formula is as follows:average fluorescence intensity = total fluorescence intensityof the area/the area.

2.17. Locomotor Capacity Assessment. The locomotor capac-ity of hind limbs of all rats was assessed by Basso, Beattie,and Bresnahan (BBB) locomotor rating scale. 0 means nomotor activity, and 21 means normal motor activity. Thementioned results were observed by two independentresearchers at 1 day before and 1, 7, 14, 21, and 28 days afterinjury.

2.18. Paw Withdraw Thermal Latency (PWTL). On days 0, 7,14, 21, and 28 after injury, the thermal sensitivity of rats wasdetected by PWTL. The radiant heat source (TechmanCompany, China) was placed in the middle of the posteriorsurface of the rat sole. After thermal stimulation, the rats’hind paw moved or 20 s later, the thermal stimulator wouldautomatically close to avoid tissue damage, and the painthreshold time was recorded. The effects of 50% radiant heatintensity and 6-8 s heat block latency on the normal painthreshold were observed.

2.19. Hind Leg Neurophysiological Experiment. After 1month of treatment, rats in each group underwent theexperiment. In brief, 4% sodium pentobarbital (50mg/kg)were adopted to anesthetize animals. Transcranial electricalstimulation was applied with pulse, and a SEN-7301 con-stant current isolation stimulator (Nihon Kohden Corp.,Japan) was employed for 1ms at 9mV. Two 30 g stainlesssteel stimulation electrodes (Nihon Kohden Corp., Japan)were implanted subcutaneously into the hind limbs of ratsto record motor evoked potentials (MEPs) of rat hind limbsuntil we obtained 3~5 stable and repeatable potentials, andwe continuously recorded the amplitude of MEP for 3 times.

2.20. Statistical Analysis. SPSS 21.0 statistical software wasused (IBM Corp., Armonk, NY, USA). Data were comparedby performing two-tailed Student’s t-test or one-way analy-sis of variance and Tukey’s post hoc test. p < 0:05 was con-sidered to be statistically significant.

3. Results

3.1. Characterization of Exo and ExomiR-494. As mentionedabove, we successfully isolated MSCs. Flow cytometryshowed that the isolated BMSCs highly expressed CD29(99.9%), CD90 (99.2%), and low expressed CD45 (0.37%),which were in accordance with the characteristics of ratBMSCs (Figure 2(a)). Next, we extracted considerable Exo

Table 2: Primers used for RT-qPCR.

Gene Sequence (5′ to 3′)

miR-494Forward CATAGCCCGTGAAACATA CACG

Reverse GTGCAGGGTCCGAGGT

U6Forward CGCTTCGGCAGCACATATACTAReverse GCGAGCACAGAATTAATACGAC

5Oxidative Medicine and Cellular Longevity

from the supernatant of rat bone marrow MSCs by the kitmethod and compared Exo with the subsequent construc-tion of ExomiR-494. First, we observed Exo/ExomiR-494 underTEM by negative staining, and TEM results showed thatmany Exos and ExomiR-494 (the yellow arrow indicates Exosor ExomiR-494) had a typical saucer-like structure with a sizeof approximately 30–200 nm (Figure 2(b)), and NTA alsoshowed that the diameters of Exo and ExomiR-494 were con-centrated at about 150nm (Figure 2(c)), which showed thatthe incorporation of miR-494 did not affect the diameter ormorphology of Exo. Moreover, the WB band also showedthat miR-494 did not affect the expression of Exo-labeledproteins CD9 and CD63 (Figure 2(d)).

3.2. Exosome Loading miR-494 Has Good Loading Rate andStability. According to existing studies [21], we loadedmiR-494 into exosomes by Exo-Fect. The results of absolutequantitative RT-PCR showed that the exosomes of the Exo-Fect group contained more miR-494 molecules, with thenumber of about 2:26 ∗ 1014/μg exosomes (Figure 3(a)),and the sample loading rate/transfection efficiency of miR-

494 is about 38.24% (Figure 3(b)). Interestingly, the numberof molecules in the Control group (untransfected) also had1:12 ∗ 1011/μg exosomes, which may be due to the fact thatexosomes can also actively take up some miRNAs in verysmall amounts (Figure 3(b)).

Furthermore, in order to test the stability of ExomiR-494

in the circulatory system, we treated naked miR-494 andExomiR-494 with RNase A. The results of RT-PCR showedthat the naked miR-494 was degraded gradually with time,and the residual amount of miR-494 was less than 15% at90min, while the residual amount of miR-494 in ExomiR-494

was still 44:8 ± 2:1% (Figure 3(c)). These results showed thatexosomes had a good drug loading effect and could effectivelyprevent the degradation of miRNAs in body fluid.

3.3. Therapeutic Effect of ExomiR-494 on DRGs In Vitro.Existing studies showed that MSC-derived pure exosomesor pure miR-494 could inhibit neuronal apoptosis [18]. AfterLPS/hypoxia treatment, the DRGs were treated with PBS,ExomiR-con, and ExomiR-494. Live/dead cell double stainingshowed that the DRG survival rate of the Control group

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6 Oxidative Medicine and Cellular Longevity

was significantly decreased, only 46.35%, while the survivalrate of the Exo-miR-494 group was as high as 80:1 ± 2:5%(Figures 4(a) and 4(b)); In addition, the Exo-miR-con groupalso had a survival rate of 58:2 ± 2:6%, which was attributedto the antiapoptotic effect of the exosome alone (Figures 4(a)and 4(b)). The WB trend of apoptotic protein was the sameas the cell survival rate (Figure 4(c)). The Caspase-3 and Baxin the Exo-miR-con group and the Exo-miR-494 group weresignificantly lower than those in the Control group(Figure 4(d)). On the contrary, the antiapoptotic proteinBcl-2 in the Exo-miR-con and Exo-miR-494 groups wassignificantly higher than that in the Control group(Figure 4(d)), because both exosome and miR-494 have anti-apoptotic effects, which may be coordinated with each otherand conform to the cell survival trend.

3.4. ExomiR-494 Can Promote the Polarity Transformation ofMacrophages In Vitro and Inhibit Inflammatory Reaction.MSC-derived exosomes can regulate the polarization ofmacrophages and reduce the inflammatory response afterSCI [24, 25]. Next, we studied the relationship between

ExomiR-494 and inflammatory response. The alveolar mac-rophages of NR8383 rats were treated with LPS/hypoxiafor 24 h and then cocultured with miR-494, ExomiR-con,and ExomiR-494 for 48 h. The expression of MI and M2markers was detected by ELISA. As revealed from theresults, ExomiR-con or ExomiR-494 treatment significantlydecreased the expression of M1 polarization-related proin-flammatory cytokines (TNF-6 and IL-α), but the differencebetween the two groups was not obvious (Figure 4(e)).Meanwhile, ExomiR-con or ExomiR-494 treatment also signifi-cantly increased the expression of M2 polarization-relatedanti-inflammatory cytokines (IL-8 and IL-10) in Figure 4(f).

3.5. ExomiR-494 Can Home into the Spinal Cord Lesion in Ratsby Tail Vein Injection. Next, animal experiments were per-formed. The DIR iodide-labeled ExomiR-494 were injectedinto SCI rats via the tail vein, and its distribution in theorgans and spinal cord was observed under the animal fluo-rescence imaging system. As revealed from the results, 3 himaging results showed that ExomiR-494 showed relativelyhigh fluorescence intensity in the lung and liver, which was

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%)

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Figure 3: Exosome loading miR-494 by Exo-Fect. (a) The absolute number of miR-494 molecules in exosomes by absolute quantitative real-time PCR. n = 3, data are represented as mean ± SD, ∗∗p < 0:01 vs. Control. (b) The loading rates of miR-494 in Exo were presented. n = 3,data are represented as mean ± SD, ∗∗p < 0:01 vs. Control. (c) Relative contents of naked miR-494 and ExomiR-494 after RNase A treatment.n = 6, data are represented as mean ± SD, ∗p < 0:05 and ∗∗p < 0:01 vs. naked miR-494.

7Oxidative Medicine and Cellular Longevity

Control Exo-miR-con

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Figure 4: Continued.

8 Oxidative Medicine and Cellular Longevity

consistent with existing research results (Figure 5(a)) [26],that is, exosomes injected into the body were mainly phago-cytized by mononuclear in the liver. The results of 24 hshowed that the fluorescence intensity of all ExomiR-494

decreased significantly, demonstrating that ExomiR-494 wereabsorbed by liver cells and nerve cells in the injured area(Figure 5(a)).

3.6. ExomiR-494 Injected by Tail Vein Has No Toxic Effect onthe Liver. According to the fluorescence images of animals(Figure 5(a)), we know that most of ExomiR-494 convergesto the lung and liver, so we conducted a toxicity test onhealthy rats after administration of ExomiR-494. The HEstaining results showed that the lung and liver of the Exo-miR-494 group showed a normal shape and contour,without obvious signs of injury (Figures 5(b) and 5(c)).Furthermore, the activities of enzymes closely related toliver function were detected. The results demonstrated thatthe activities of LDH, SOD, and ALT in rat liver afterExomiR-494 treatment were similar to those in healthy rats,indicating that the exosome has no toxic side effects on ratliver, which is an ideal miRNA vector (Figure 5(d)).

3.7. ExomiR-494 Can Reduce the Lesion Volume of the SpinalCord. Next, we evaluated the injury volume of the transversesection of the spinal cord by HE staining. At day 28 post-SCI, the lesion center of the Control group often containedtypical vacuoles with irregular shape (Figure 6(a)). Com-pared with the Control group, the central contour of Exo-miR-494 was more complete, the shape was more normal,and the damage volume was smaller (Figure 6(a)). In addi-tion, the damage degree of the Exo-miR-con group wasbetween that of the Control group and the Exo-miR-494group (Figure 6(a)).

3.8. ExomiR-494 Promotes the Regeneration of Neurofilamentin Spinal Cord Injury Rats. To determine the effect ofExomiR-494 on neurofilament regeneration, NF-H and GFAPdouble-labeled immunofluorescence were employed toevaluate neurofilament regeneration and astrocyte activation4 weeks after injury and as revealed from the results(Figure 6(b)). As a result, considerable active astrocytes wererecruited and the neurofilament disintegrated in the Controlgroup (Figure 6(b)), and the NF-positive cells in the Exo-miR-494 group were significantly higher than those in theControl group and the Exo-miR-con group (Figure 6(c));also, the GFAP-positive cells were lower than in the Controlgroup and the Exo-miR-con group (Figure 6(d)). Thus, theExo-miR-494 group rats showed significantly increased neu-rofilament and decreased astrocytes (Figure 6(d)).

At the same time, after 28 days of injury, we measuredthe expression of GAP-43 (Figure 6(e)). As a result, afterinjury, GAP-43 was upregulated in all three groups exceptthe Sham group (Figure 6(f)). Among them, the upregula-tion was the highest in the Exo-miR-494 group, up to110 ± 4:0%, and there was also a certain increase in theExo-miR-con group (Figure 6(f)), which was derived fromsimple exosomes and promoted the increase of GAP-43.

3.9. ExomiR-494 Promotes the Recovery of Behavioral Functionin Rats with Spinal Cord Injury. Lastly, we evaluated thebehavioral function of rats. To determine the effect ofExomiR-494 on the recovery of the motor function of allrats, the BBB score was used to evaluate the locomotorcapacity of all rats at each time point after injury, and asrevealed from the results, the BBB scores of rats in eachgroup increased gradually with time due to SCI rats alsohaving a certain self-restoring capacity (Figure 7(a)). Onday 28, both Exo-miR-494 group and Exo-miR-con group

IL-6 TNF-𝛼

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Figure 4: Therapeutic effect of ExomiR-494 on DRGs and NR8383 macrophages in vitro. (a) Cell survival of DRGs was measured by AOEBstaining in Control, Exo-miR-con, and Exo-miR-494 groups (the living cells are green and the dead cells are red). (b) Cell viability by cellcounting in the Control, Exo-miR-con, and Exo-miR-494 groups. n = 3, data are represented as mean ± SD, ∗p < 0:05 and ∗∗p < 0:01 vs.Control. (c) Western blot analysis of apoptosis-related proteins (Caspase-3, Bax, and Bcl-2) at 24 h in the Control group, Exo-miR-congroup, and Exo-miR-494 group. (d) Relative density of Caspase-3, Bax, and Bcl-2. n = 6, data are represented as mean ± SD, ∗∗p < 0:01vs. Control. (e) ELISA analysis for secretion of M1 polarization-related proinflammatory cytokines (IL-6, TNF-α) and (f) M2polarization-related proinflammatory cytokines (IL-8, IL-10) in LPS/hypoxia-treated NR8383 macrophages at 48 h after ExomiR-494,ExomiR-con, and PBS administrations. n = 3, data are represented as mean ± SD, ∗∗p < 0:01 vs. Control.

9Oxidative Medicine and Cellular Longevity

3 h

24 h

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intensity

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Figure 5: Therapeutic effect of ExomiR-494 on spinal cord injury in vivo. Rats were classified into Control group, Sham group, Exo-miR-congroup, and Exo-miR-494 group. (a) Fluorescence imaging of organs of Exo-miR-494 group rats at 3 h and 24 h after intravenous injectionwas illustrated. (b) HE staining of lung in the health group and Exo-miR-494 group. The rats in the health group were fed normally withoutany injury or treatment. Scale bar = 50 μm. (c) HE staining of liver in health group and Exo-miR-494 group. Scale bar = 50μm. (d) Enzymeactivities of LDH, SOD, and ALT in the liver of health group and Exo-miR-494 group rats. n = 6. (e) The relative expressions of miR-494 ininjured spinal cord at 48 h after ExomiR-494-con, ExomiR-494, and PBS treatment by RT-PCR. n = 6, data are represented as mean ± SD,∗∗p < 0:01 vs. Control.

10 Oxidative Medicine and Cellular Longevity

animals showed significant functional recovery, of which theaverage score of the Exo-miR-494 group was 15:8 ± 0:7 andthat of Exo-miR-con animals was 12:7 ± 0:5 (Figure 7(a)).The average score of the Control group was lower, whichwas only 11:2 ± 0:65 (Figure 7(a)). So, the motor functionalrecovery of the Exo-miR-494 group was better than thoseof the Exo-miR-con group and Control group.

To further analyze thermal sensitivity by PWTL, on thefirst day before injury, all rats showed almost the same andnormal PWTL, with an average of about 6:8 ± 0:5 s(Figure 7(b)). One week after spinal cord injury, PWTL ofrats in each group was more than 20 s due to severe trauma(Figure 7(b)). Then, the PWTL of rats in each groupdecreased gradually, and the most obvious decrease was in

Control Sham Exo-miR-con Exo-miR-494

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Figure 6: ExomiR-494 can reduce the lesion volume of spinal cord and promote the regeneration of neurofilament. (a) On the 28th day afterinjury, the transverse section of spinal cord in each group was stained with HE. Scale bar = 100μm. (b) Immunofluorescence images showingthe expressions of neurofilament (NF, green) and astrogliosis (GFAP, red) in longitudinal section of the spinal cord lesion at 4 weeks in eachgroup. Scale bar = 200 μm or 50μm. (c) Relative average fluorescence intensity of NF (%) in each group rats by ImageJ software. The averagefluorescence intensity in Sham group was set to 100%. n = 6, data are represented as mean ± SD, ∗p < 0:05 and ∗∗p < 0:01 vs. Control.(d) Relative average fluorescence intensity of GFAP (%) in each group of rats. The average fluorescence intensity in Control group wasset to 100%. n = 6, data are represented as mean ± SD, ∗p < 0:05 and ∗∗p < 0:01 vs. Control. (e) Expression of GAP-43 in rats of eachgroup after 28 days of SCI. (f) Relative density of GAP-43 by ImageJ software. n = 6, data are represented as mean ± SD, ∗p < 0:05 and∗∗p < 0:01 vs. Control.

11Oxidative Medicine and Cellular Longevity

BBB

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12 Oxidative Medicine and Cellular Longevity

the Exo-miR-494 group, reaching 12:7 ± 0:4 s on the 28thday, 16:5 ± 0:8 s in the Exo-miR-con group, and 17:5 ± 0:3 sin rats of the Control group (Figure 7(b)).

3.10. ExomiR-494 Promotes the Recovery of Neuroelectrophysiologyin Spinal Cord Injury Rats. 4 weeks after injury, we conductedneuroelectrophysiological experiments (Figure 7(c)). Asshown in Figures 7(d) and 7(e), the MEPs in hind limbs ofall rats showed that there was an obvious one-way wavein the Exo-miR-494 group, and the average amplitude(184 ± 4 μV) was significantly larger than that in theExo-miR-con (34 ± 5 μV) or Control groups (14 ± 5 μV).Therefore, the average amplitude of MEP in the Controlgroup was very small, which was due to the ineffectiverecovery of neurophysiological function.

4. Discussion

SCI is capable of causing severe motor and sensory disordersin patients, and no effective treatment has been applied tothe clinic thus far [27]. It is increasingly evidenced thatmany miRNAs are involved in the pathogenesis or recoverymechanism. To be specific, miR-135a-5p can promote therecovery of spinal cord injury by mediating ROCK pathway[28], and low-level miR-130a-3p can activate the IGF-1/IGF-1R pathway to relieve pathological pain after spinal cordinjury [29].

miR-494 refers to a tumor suppressor inhibiting the pro-liferation and colony formation of tumor cells [30]. miR-494can reduce autophagy and apoptosis of PC-12 cells inducedby LPS by targeting IL-13 in vitro [18]. Besides, miR-494 canalso regulate the PTEN/Akt/mTOR pathway, thereby inhi-biting neuronal apoptosis after SCI in rats [22, 31]. Further-

more, as reported by our previous research, SIRT1 caninhibit apoptosis after spinal cord injury via miR-494 [32].

Stem cell transplantation has been investigated in tissueregeneration for years; however, it still has some limitations[33, 34]. Over the past few years, exosomes, a novel type ofintercellular communication device, have been employed asa good biological carrier for local or systemic small RNAdelivery for treating stroke or spinal cord injury and othercentral nervous system diseases. The mentioned exosomesplay an active role in tissue regeneration, while acting asthe carriers of genetic materials (e.g., mRNAs, miRNAs,and lncRNAs), which can be used as gene transfer systemsfor alternative cell therapy. Here, ExomiR-494 was constructedby chemical transfection, and the therapeutic effect of Exo-miR-494 on SCI was studied in depth.

First, miR-494 was loaded into the exosome effectivelyby chemical transfection, and it exhibited high loading effi-ciency, about 38% loading rate, complying with the existingresults [21]. The amount of exosomes secreted in vivo is verysmall, so convenience was provided for the mass productionof ExomiR-494 by chemical transfection, which is considered ahigh loading efficiency method. Besides, exosomes exhibitexcellent encapsulation and antidegradation properties,capable of effectively preventing the degradation of targetmiRNA in vivo environment, which provides the possibilityfor the tail vein administration of ExomiR-494. It is generallyknown that innate immune cells (i.e., microglia and astro-cytes) and infiltrating leukocytes (i.e., macrophages andneutrophils) are activated after SCI, thereby leading toinflammatory cascade. The mentioned inflammatory cellsrelease various neurotoxins, proinflammatory cytokines,chemokines, free radicals, excitotoxic amino acids, nitricoxide, and others, creating a very unfavorable microenviron-ment for the regeneration of neurons [35]. In addition, glial

The a

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ge am

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-494

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Figure 7: ExomiR-494 promotes functional recovery in SCI rats. (a) BBB scores of rats in Control group, Sham group, Exo-miR-con group,and Exo-miR-494 group. n = 6, data are represented as mean ± SD, ∗p < 0:05 and ∗∗p < 0:01 vs. Control. (b) The PWTL of rats in Shamgroup, Control group, Exo-miR-con group, and Exo-miR-494 group. n = 6, data are represented as mean ± SD, ∗p < 0:05 and ∗∗p < 0:01vs. Control. (c) Schematic diagram of neuroelectrophysiology. (d) MEPs of rats in Sham group, Control group, Exo-miR-con group, andExo-miR-494 group after 4 weeks of treatment (the average of the three times was collected into a line graph, and the red arrow markedthe stimulus artifact). (e) The average amplitude in Sham group, Control group, Exo-miR-con group, and Exo-miR-494 group after4 weeks of treatment. n = 6, data are represented as mean ± SD, ∗p < 0:05 and ∗∗p < 0:01 vs. Control.

13Oxidative Medicine and Cellular Longevity

scar imposes a major obstacle on axonal regeneration. Afterspinal cord injury, injured fibroblasts invade the injured areaand then secrete considerable extracellular matrix (e.g., typeIV collagen, fibronectin, and laminin), constituting the maincomponent of the glial scar [35, 36]. Wang et al. reportedthat miR-494 can inhibit the proliferation and synapticremodeling of spinal reactive astrocytes in SCI rats by acti-vating the Nogo/NgR signaling pathway [19]. Reactive astro-cytes are recognized as the pathological markers of centralnervous system damage (e.g., Parkinson’s disease, Alzhei-mer’s disease, and spinal cord injury) [37]. Reactive astro-cytes are capable of secreting considerable extracellularmatrix and forming glial scar, thereby leading to limitednerve repair and axonal degeneration [38, 39]. Moreover,glial scar formation is capable of stimulating the productionof GFAP, which activates the RhoA signal, causes the growthcone to collapse, and inhibits axon regeneration [40].

In this study, as revealed from NF and GFAP immuno-fluorescence results, ExomiR-494 can effectively downregulatethe expression of GFAP, as well as promote the increase ofthe neurofilament. Indeed, the mentioned positive resultsreveal the important role of miR-494 for treating spinal cordinjury. Over time, the BBB score of ExomiR-494 group wassignificantly higher than those of other groups, demonstrat-ing that the motor function of rats had been significantlyimproved. In addition, to explore the effect of ExomiR-494

on the electrophysiological function of the hind limbs ofSCI rats, the electrophysiological study was conducted onthe hind limbs of rats in 4 weeks. Complying with the existingresults, ExomiR-494 is capable of effectively facilitating therecovery of the neuroelectrophysiological function of SCI rats.

5. Conclusion

In brief, the results showed that the exosome is an excellentmiRNA vector, the tail vein injection of ExomiR-494 canupregulate the expression of miR-494, improve the localimmune environment, and inhibit neuronal apoptosis andrelease of proinflammatory factors, thereby promoting theregeneration of the neurofilament and the recovery ofbehavioral function.

Data Availability

The [experimental data] data used to support the findings ofthis study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Supplementary Materials

Supplementary 1. Supplement 1: instruction manual of theGS exosome isolation reagent kit.

Supplementary 2. Supplement 2: Exo and Exo-miR-494 neg-ative staining steps for transmission electron microscope.

Supplementary 3. Supplement 3: real-time polymerase chainreaction (RT-PCR) experimental steps.

Supplementary 4. Supplement 4: ALT test kit instructions.

Supplementary 5. Supplement 5: LDH test kit instructions.

Supplementary 6. Supplement 6: SOD test kit instructions.

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